The goal of tissue engineering is to augment, replace, or restore complex human tissue function by combining synthetic and living components in appropriate configurations and environmental conditions. There are three key aspects to consider in any tissue-engineered construct - the cells, the matrix or biomaterial construct, and cell-material interactions. Although increasing numbers of research groups are developing techniques to control cell growth in artificial matrices, few have investigated cell-matrix interactions and the evolving mechanical properties of three-dimensional (3-D) mechanically-stimulated engineered tissues. The primary limitation has been inadequate imaging technology for high-resolution, real-time, non-invasive imaging deep within scattering tissue. The 3-0 arrangement of cell populations strongly influences the way in which cells dynamically respond within the engineered tissue. Optical imaging techniques that permit deep-tissue 3-D imaging offer the opportunity to non-invasively track the formation of engineered tissues. This project will integrate and apply two complementary state-of-the-art optical imaging techniques, optical coherence tomography and multi-photon microscopy, which are capable of performing these imaging tasks. Both optical techniques utilize the same laser source and will be integrated in a single microscope to investigate dynamic cell-matrix interactions and the evolving mechanical properties of two model engineered tissues. These optical imaging techniques will permit high-resolution, real-time, deep-tissue imaging in 3-D to non-invasively and non-destructively track changes in the tissue formation of 3-D blocks of cardiac myocytes and vascular structures composed of fibroblasts and endothelial cells. We will use these imaging capabilities and biomolecular focal adhesion assays to determine the influence that the 3-D microenvironment and dynamic mechanical forces have on the growth, organization, and mechanical properties of these model engineered tissues. The development and application of this unique investigative microscope will improve our understanding of cell function and tissue dynamics in 3-D mechanically-stimulated culture environments, enabling generation of engineered tissues with increasingly complex functionality.